Overall, we evidenced that CK2 inhibitor CX-4945 impinge on mediators of signaling pathways and biological processes essential for primary AML cells survival and chemosensitivity, reinforcing the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy

Overall, we evidenced that CK2 inhibitor CX-4945 impinge on mediators of signaling pathways and biological processes essential for primary AML cells survival and chemosensitivity, reinforcing the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy. Test and p-value < 0. 05 was considered statistically significant. found differentially modulated in HL-60 and OCI-AML3 cells, respectively. Despite regulated phosphopeptides belong to proteins Rupatadine Fumarate involved in multiple biological processes and signaling pathways, most of these perturbations can be explain by direct CK2 inhibition rather than off-target effects. Furthermore, CK2 substrates regulated by CX-4945 are mainly related to mRNA processing, translation, DNA repair, and cell cycle. Overall, we evidenced that CK2 inhibitor CX-4945 impinge on mediators of signaling pathways and biological processes essential for primary AML cells survival and chemosensitivity, reinforcing the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy. Test and p-value < 0.05 was considered statistically significant. We Rupatadine Fumarate also applied a fold-change (treated vs. control) threshold of 1 1.5 (|FC| 1.5) to define the down- and up-regulated phosphopeptides and proteins. In HL-60 cells 275 phosphopeptides on 224 proteins were significantly modulated, while in OCI-AML3 cells the number was almost 5-fold higher with 1324 on 847 proteins (Figure 2A, Table S1). In both cellular contexts, treatment with CX-4945 elicited a global decrease of protein phosphorylation, based on the distribution of down- and up-regulated phosphopeptides in Volcano plots (Figure 2A). On the contrary, proteomic analysis indicated that in both cell lines Rupatadine Fumarate CK2 inhibition showed no bias towards the protein down-regulation (Figure 2B, Table S2). Actually, proteome analysis evidenced that changes in phosphorylation upon CX-4945 treatment were mostly independent of protein abundance, since only eight down-regulated proteins (two in HL-60 cells and six in OCI-AML3 cells) had phosphorylation sites significantly inhibited (Figure 2B). Those proteins were not considered as differentially phosphorylated after CK2 inhibition, and consequently, were not included in the functional interpretation of the phosphoproteomic profiles. Open in a separate window Figure 2 Phosphoproteomic and proteomic profile of human AML cells treated with the CK2 inhibitor CX-4945. Volcano plots of quantified (A) phosphopeptides and (B) proteins from HL-60 and OCI-AML3 cells after treatment with 5 M CX-4945 during 8 h. Red points indicate those phosphopeptides/proteins that met statistical significance cut-off (|FC| 1.5, p-value < 0.05). Additionally, black points indicate those phosphopeptides with decreased phosphorylation due to the reduction of the corresponding protein abundance in proteomic analysis (down-regulated proteins are also indicated in black). In summary, after normalization with the proteome dataset a total of 273 and 1310 significantly modulated phosphopeptides were identified in HL-60 and OCI-AML3 cells, respectively (Figure 1B and Figure 2A). Remarkably, such difference indicates that CX-4945 has a more pronounced effect over the CK2-dependant signaling in OCI-AML3 cells, which suggests that the molecular perturbations induced by this inhibitor could rely on the AML cellular background. However, CX-4945 had a similar dose-dependent inhibitory effect on HL-60 and OCI-AML3 cells proliferation (Figure S1A), suggesting that despite the divergence concerning the molecular impact of protein kinase CK2 inhibition, there is no differential sensitivity of AML cells towards the overall antiproliferative effect of CX-4945. 3.2. Enrichment Analysis of Differentially Modulated Phosphoproteins For better understanding of putative biological processes perturbed after CK2 inhibition in AML cells, the differentially modulated phosphoproteins were classified in terms of their biological functions using the information from the GO database [33,34]. Analysis was performed using DAVID web-based tool and GO terms list was further submitted to REViGO for redundancy reduction [35,36,37]. Significantly represented biological processes in both phosphoproteomics profiles include mRNA processing, regulation of viral process and protein sumoylation (Figure 3). Moreover, phosphorylation sites differentially modulated in HL-60 are located on phosphoproteins related to mRNA splicing, cellular response to DNA damage and ribosome biogenesis, while in OCI-AML3 covalent chromatin modification, nuclear transport, legislation of cell proliferation Rabbit Polyclonal to RAB41 and gene appearance are considerably represented (Amount 3). Of be aware, apoptotic signaling pathway was just defined as enriched in OCI-AML3 cells significantly. Consistently, our outcomes and previous research have got evidenced that HL-60 cell series shows refractoriness to CX-4945 induced apoptosis (Amount S1B), probably due Rupatadine Fumarate to the lack of p53 protein (HL-60 cells are p53 null) and the low CK2 protein level and activity compared to various other AML cell lines [51]. In such research it was showed that CK2 inhibition not merely sets off apoptotic cell loss of life in AML cell lines, however Rupatadine Fumarate in freshly isolated blasts from AML sufferers [51] also. Open up in another window Amount 3 Enrichment evaluation for differentially modulated phosphoproteins in HL-60 and OCI-AML3 cells treated with CX-4945. Natural processes considerably represented in phosphoproteomic profile had been discovered using annotations from Move data source. The p-worth of improved Fisher Exact Check from DAVID is positioned in square mounting brackets. Lately, another phosphoproteomic research in non-small cell lung cancers (NSCLC) cell series NCI-H125 using the clinical-grade.